The tumor-suppressive functions of the human INK4A locus P.Mathijs Voorhoeve, Reuven Agami  Cancer Cell  Volume 4, Issue 4, Pages 311-319 (October 2003) DOI: 10.1016/S1535-6108(03)00223-X

Figure 1 Inactivation of p53 and pRb in cellular transformation of primary human fibroblasts A: Schematic drawing of the transformation protocol of human primary BJ fibroblast using overexpression of hTERT, SV40 Large and small t antigens (LT and st), and H-RASV12. LT inhibits the Rb and p53 pathways. B: Stable suppression of pRb and p53 expression in primary human fibroblasts. BJ-ET cells were infected with the indicated retroviral siRNA vectors (pRetroSuper (pRS)-Hyg) and selected for 10 days with hygromycin. Subsequently, polyclonal pools of cells were infected with a mixture of retroviruses expressing oncogenic RASV12 and st and selected with blasticidin. The efficiency of st infection was controlled with GFP and was consistently between 50%–60%. After seven days of selection, cells were harvested and protein expression was analyzed on SDS-PAGE with the indicated antibodies. pRb protein is indicated by the vertical bar and background staining is indicated by an asterisk. C: The same cell populations from B were passed through a 3T3 protocol in which at every passage, the relative increase of the cell number was monitored. D: The same cell population as in B was plated in soft agar to test anchorage-independent growth capability. Visible colonies were counted after three weeks. E: Six aliquots of 2 × 10E6 BJ-ET cells from B were injected into the flanks of three athymic nude mice, and the number of visible tumors was noted after four weeks. Cancer Cell 2003 4, 311-319DOI: (10.1016/S1535-6108(03)00223-X)

Figure 2 Specific inhibition of expression of p14ARF and p16INK4A A: Overview of the INK4A locus. The open reading frames of p14ARF (light gray) and p16INK4A (dark gray) are indicated. Dashes above the unique exons illustrate the position of the siRNA oligos used to target the respective mRNAs. B: Stable suppression of p16INK4A and p14ARF expression by retroviral transduction. pRS-Hygro retroviruses containing the indicated siRNA constructs were used to infect HeLa cells. After selection for one week, protein expression was analyzed by immunoblot with the indicated antibodies. Equal loading control is shown with several background bands (bg). Cancer Cell 2003 4, 311-319DOI: (10.1016/S1535-6108(03)00223-X)

Figure 3 Growth-inhibiting function of INK4A genes A: Schematic depiction of the experiment. Cells are transduced with the siRNA-expressing GFP vectors, and the percentage of GFP-positive cells was determined later by flow cytometry. Increase in the GFP-positive population in time indicates a relative growth advantage whereas decrease indicates a relative growth disadvantage. B and E: Primary human BJ-ET cells, that were first transduced with RS-Hyg-53#2, RS-Hyg14#1, RS-Hyg16#4, or control RS-Hyg and selected (BJ53, BJ14, BJ16, or BJET, respectively), were transduced with the indicated knockdown-GFP vectors and monitored for a period of 10–20 days. In E, right panel, data from B-left and E-left is copied to allow direct comparison in the same scale. C: MCF7 cells were infected with the indicated retroviruses and cumulative growth advantage was monitored over 19 days. D: RNA from BJ-ET cells, stably transduced with the indicated vectors, was collected and subjected to RT-PCR. Relative band intensities of the specific p14ARF product (marked with an arrow) are indicated by the bar histogram. As control, RT-PCR for CDK4 was performed. F: p16 expression was analyzed in BJ-cells expressing the indicated knockdown constructs. For comparison with cells with elevated p16 levels, BJET cells expressing Ras were included (lane 1). Cancer Cell 2003 4, 311-319DOI: (10.1016/S1535-6108(03)00223-X)

Figure 4 p16INK4A and p53 collaborate to protect from oncogenic transformation A: BJ-ET cells were infected with various knockdown- or LT-expressing viruses and subsequently infected with Ras and st and analyzed as in Figure 1B. B: BJ-ET cells were transduced with a RASV12 vector (lane 4) or control vector (lane 3) and selected for a week. Cell extracts were analyzed with the indicated antibodies. For comparison, p14ARF expression was monitored in HeLa cells transduced with control (lane 2) or the p14ARF siRNA vector (lane 1). C: Growth curves of the indicated knockdown BJ-ET-st-RASV12 cells. D: Immunoblot analysis of whole-cell lysates from the cells in C before and after RASV12 transduction. Short exposure is indicated by s.e. E: Soft agar assay with the cells from C. F: Tumorigenicity in nude mice was assayed as in Figure 1E. G: A schematic model showing the growth and tumor-suppressive functions of the INK4A genes. Cancer Cell 2003 4, 311-319DOI: (10.1016/S1535-6108(03)00223-X)